ABSTRACT Hematopoietic stem cell (HSC) transplants are used to treat several malignant and non-malignant hematological diseases. Unfortunately, even after decades of research, HSCs cannot be well maintained or expanded ex vivo, which has left many patients without a sufficient source of potentially life-saving HSCs. Even short culture times in optimized conditions are deleterious to HSCs, limiting opportunities for HSC expansion or gene editing. In preliminary studies, we established a culture system that supports sustained ex vivo HSC growth. In this system, ten purified HSCs can be expanded into ~104 hematopoietic stem and progenitor cells over a 10-day period, without any loss of serial long-term multilineage reconstituting activity. The development of this culture system was based on our discovery that HSCs have lower rates of protein synthesis than other blood cells, and that modest increases in protein synthesis impair HSC function. We determined that increased protein synthesis impairs HSCs by reducing proteome quality. Strikingly, HSCs exhibit a massive increase in protein synthesis in vitro that reduces proteome quality and disrupts protein homeostasis (proteostasis). We thus sought a way to circumvent this collapse in proteostasis when HSCs are removed from their in vivo environment so as to allow their maintenance and expansion for therapeutic applications. The heat shock response is the principal pathway that responds to proteotoxic stress in the cytoplasm. The master regulator of the heat shock response is Heat shock factor 1 (Hsf1). Under conditions of proteotoxic stress, Hsf1 induces transcription of heat shock proteins that coordinate protein folding, trafficking and degradation to promote proteostasis and cell survival. We have established that Hsf1 promotes ex vivo HSC maintenance, as conditional deletion of Hsf1 significantly exacerbates HSC loss in vitro. Furthermore, we identified small molecules that enhance Hsf1 activation within cultured HSCs and these small molecules significantly enhance ex vivo HSC growth and maintenance in a Hsf1-dependent manner. Based on these data, we hypothesize that proteotoxic stress impairs HSC self-renewal and contributes to HSC depletion in vitro, and interventions that increase Hsf1 activity can promote ex vivo HSC growth by enhancing proteostasis capacity. In Aim 1 we will use a suite of new technologies to test how gain and loss of Hsf1 activity influence proteostasis within HSCs. In Aim 2 we will culture adult mouse and human HSCs in the presence of Hsf1 activators and test if these treatments enable HSC expansion by performing limiting dilution transplants. In Aim 3 we will determine how ex vivo growth influences the identity of HSCs using cell surface profiling and single cell RNA-sequencing. Our studies represent a new approach for promoting ex vivo HSC growth by activating the heat shock response and enhancing proteostasis capacity. Identifying a modality for HSC maintenance and expansion holds enormous therapeutic potential for patients with diverse hematopoietic disorders.